MEK kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxin-induced apoptosis - PubMed (original) (raw)

MEK kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxin-induced apoptosis

C Widmann et al. Mol Cell Biol. 1998 Apr.

Abstract

MEK kinase 1 (MEKK1) is a 196-kDa protein that, in response to genotoxic agents, was found to undergo phosphorylation-dependent activation. The expression of kinase-inactive MEKK1 inhibited genotoxin-induced apoptosis. Following activation by genotoxins, MEKK1 was cleaved in a caspase-dependent manner into an active 91-kDa kinase fragment. Expression of MEKK1 stimulated DEVD-directed caspase activity and induced apoptosis. MEKK1 is itself a substrate for CPP32 (caspase-3). A mutant MEKK1 that is resistant to caspase cleavage was impaired in its ability to induce apoptosis. These findings demonstrate that MEKK1 contributes to the apoptotic response to genotoxins. The regulation of MEKK1 by genotoxins involves its activation, which may be part of survival pathways, followed by its cleavage, which generates a proapoptotic kinase fragment able to activate caspases. MEKK1 and caspases are predicted to be part of an amplification loop to increase caspase activity during apoptosis.

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Figures

FIG. 1

FIG. 1

UV induces rapid phosphorylation of endogenous MEKK1 in HEK293 cells, followed by the cleavage of MEKK1 into smaller fragments. (A) HEK293 cells were irradiated or were not irradiated with UV (100 J/m2) and incubated for 16 h in DMEM containing 0.1% serum. The cells were then lysed and subjected to Western blot analysis with the MEKK1 NH2-terminus-specific antibody 96-001. The positions of full-length MEKK1 and the UV-generated NH2-terminal fragment (fragment B) are indicated. (B) The cells were treated with UV (100 J/m2) and incubated for the indicated times in DMEM containing 0.1% serum. (Top panel) Cell lysates were analyzed as described in the legend to Fig. 1A. For clarity, only the portions of the gel containing the 196-kDa MEKK1 protein and the immunoreactive MEKK1-derived fragment are shown. (Middle panel) Activation of the JNK pathway was measured with Sepharose-bound GST-Jun as a substrate. (Bottom panel) Propidium iodide-stained nuclei were analyzed with a fluorescence-activated cell sorter (46). The percentage of nuclei with an altered shape (apoptotic nuclei) was plotted as a function of time. (C) The cells were treated as in panel B. The kinase activity of the endogenous MEKK1 protein in response to UV-C irradiation was measured as described in Materials and Methods. The activation of MEKK1 in response to UV-C irradiation temporally correlated with its gel shift and with the activation of the JNK pathway.

FIG. 2

FIG. 2

The gel-shifted form of MEKK1 corresponds to an active phosphorylated kinase. (A) Cell lysates from HEK293 cells were transfected with 1 μg of MEKK1.cp4, carrying the HA-tagged full-length MEKK1, immunoprecipitated with antibody 12CA5, and incubated for 45 min with or without PP-2A at 37°C as described in Materials and Methods. The immunoprecipitates (IP) were then subjected to Western blot analysis with antibody 96-001. Arrows indicate the positions of the nonshifted and shifted forms of MEKK1. (B) Cell lysates from MEKK1-transfected cells were immunoprecipitated and incubated with PP-2A at 4 or 37°C or left untreated as described in panel A. After several washes, the immunoprecipitates were incubated with recombinant SEK1 K→M substrate and [γ-32P]ATP. Phosphorylated proteins were resolved by SDS-PAGE. The identity of the protein indicated by an asterisk is unknown. Note that when the immunoprecipitates were incubated with PP-2A at 4°C and washed, SEK1 K→M phosphorylation still occurred, showing that the absence of SEK1 K→M phosphorylation by immunoprecipitates treated with PP-2A at 37°C was not due to residual PP-2A activity that would not have been eliminated during the washing steps.

FIG. 3

FIG. 3

Genotoxins induce the cleavage of MEKK1. HEK293 cells were either left untreated, stimulated with UV at 100 J/m2, or incubated with 50 μM cisplatin, 100 μM etoposide, or 30 μM mitomycin in DMEM containing 0.1% fetal bovine serum. After 18 h, the cells were lysed and subjected to Western blot analysis with antibody 96-001. For clarity, only the unphosphorylated and gel-shifted full-length MEKK1 proteins and the NH2-terminal MEKK1-derived fragment (fragment B) are shown.

FIG. 4

FIG. 4

MEKK1 is involved in genotoxin-induced apoptosis. (A) HEK293 cells were transfected with 4 μg of empty pcDNA3 vector or pcDNA3 containing wild-type MEKK1 (plasmid MEKK1.dn3) or the MEKK1 K(1253)→M mutant [plasmid MEKK1(−).dn3]. After 18 h, the cells were lysed and the MEKK1 proteins were immunoprecipitated with antibody 12CA5, recognizing the NH2-terminal HA tag. The immunoprecipitates were then analyzed by Western blotting (WB) with antibody 12CA5. Alternatively, the JNK activity of the cell lysates was determined as described in Materials and Methods. Despite similar levels of expression, only the wild-type MEKK1 protein was able to induce the activity of the JNK pathway. (B) Clones of HEK293 cells stably transfected with pcDNA3 (clones V1 and V4) or pcDNA3 expressing the kinase-inactive MEKK1 K(1253)→M protein [plasmid MEKK1(−).dn3] (clones 7 and 11) were lysed, and the expression of MEKK1 was determined by Western blot analysis with antibody 12CA5. The positions of the full-length kinase-inactive MEKK1 and fragment A are indicated (see the text for details about the generation of fragments A and B). (C) The clones shown in panel B were stimulated as described in the legend to Fig. 3. Apoptotic cells were scored after acridine orange staining (41). Data are the mean ± standard error of the mean for duplicate determinations. (D) Clone 11 (described in panel B) was incubated with etoposide or irradiated with UV as described in Fig. 3. The cells were lysed and then analyzed by Western blotting with anti-HA antibody 12CA5. The positions of the full-length kinase-inactive MEKK1 and fragment A are indicated.

FIG. 5

FIG. 5

p35 and CrmA inhibit MEKK1-induced DNA fragmentation in HEK293 cells. Cells were transfected with 0.5 μg of MEKK1.cp4 alone or in combination with 2 μg of either p35.cp_ or CrmA.cp_. Two days later, the cells were stained for MEKK1 expression and for DNA fragmentation. (A) Nomarski views (magnification, ×40) of HEK293 cells transfected with MEKK1 or with MEKK1 and p35 and overlaid with fluorescent staining for MEKK1 expression (red staining) and for DNA fragmentation (green staining). (B) Views (magnification, ×160) of HEK293 cells transfected with MEKK1 alone or in combination with either CrmA or p35. (Left panels) Nomarski views. (Middle panels) Fluorescent staining for MEKK1 expression (red staining) and DNA fragmentation (green staining). (Right panels) Fluorescent staining for MEKK1 expression. In these last views, an exposure longer than that in the middle panels was used to visualize the localization of endogenous human MEKK1. The arrows indicate granular cytoplasmic localization, while the arrowheads indicate nuclear localization. (C) Quantitation of the percentage of MEKK1-transfected cells, in the presence or in the absence of the indicated proteins, that showed DNA fragmentation. The numbers in the columns indicate the number of cells transfected with MEKK1 and counted on at least four coverslips from at least two different experiments.

FIG. 6

FIG. 6

CrmA and p35 inhibit the generation of a MEKK1-derived kinase-active cleavage product. Cells were transfected as described in the legend to Fig. 5. (A) Western blot analysis of lysates with antibodies 12CA5 and 95-012. The immunoreactive proteins were detected by ECL. Fragments A, B, C, and D correspond to MEKK1 cleavage products, and the band marked with an asterisk may correspond to a dimer of fragment D (see the text). (B) A kinase-inactive MEKK1 mutant (MEKK1 K→M) is not cleaved into fragments B and C. Cells were transfected with 1 μg of vector alone (pcDNA3), HA-tagged MEKK1 in pcDNA3 (plasmid MEKK1.dn3), or HA-tagged MEKK1 K(1253)→M in pcDNA3 [plasmid MEKK1(−).dn3]. At 18 h after transfection, the cells were lysed and the presence of MEKK1 species was detected by Western blot analysis with antibody 12CA5. The positions of fragments A and B and full-length MEKK1 are indicated. (C) In vitro SEK1 K→M phosphorylation assay performed on cell lysates immunoprecipitated with the indicated antibodies. The positions of MEKK1, fragments C and D, and SEK1 K→M are indicated.

FIG. 7

FIG. 7

Mutation of the mouse MEKK1 sequence 871DTVD874 blocks p35-inhibited MEKK1 cleavage. (A) Schematic representation of the HA-tagged mouse MEKK1 protein showing the regions (the numbers correspond to the positions of the amino acids) used to generate the indicated antibodies. Also shown is the sequence (one-letter code) between amino acids 853 and 888 where the tetrapeptides DEVE and DTVD (in bold type) are replaced with alanine residues in mutants DEVE→A and DTVD→A, respectively. (B) Western blot analysis with the antibodies shown in panel A of lysates derived from HEK293 cells transfected with 0.5 μg of pcDNA_3.cp4, MEKK1.cp4, DEVE_A.cp4, or DTVD_A.cp4. Letters A to D indicate the same cleavage products as those shown in Fig. 6A. (C) Full-length activated MEKK1 and fragment C have similar SEK1 K→M phosphorylation activities. HEK293 cells were transfected with 4 μg of MEKK1 in pcDNA3 (MEKK1k.dn3 plasmid) or fragment C in pcDNA3 (G875.dn3 plasmid). At 18 h after transfection, 5 mg of cell lysate proteins was immunoprecipitated with antibody 12CA5 (recognizing the NH2-terminal HA tag). Serial dilutions of the immunoprecipitates (1:1, 1:2, 1:4, and 1:8) were then analyzed by Western blotting with antibody 95-012 (recognizing the COOH terminus of MEKK1) or analyzed for their ability to phosphorylate the SEK1 K→M substrate. (D) Schematic representation of p35-inhibited and p35-insensitive cleavage in the mouse MEKK1 protein. Letters A to D indicate the names of the cleavage products. The molecular masses were calculated from the migration of the markers in at least two different experiments, such as the one presented in panel B.

FIG. 8

FIG. 8

The DTVD sequence in MEKK1 is a caspase-3-like cleavage site. Wild-type MEKK1 and the DTVD→A mutant were translated in vitro as described in Materials and Methods. (A) In vitro-translated wild-type MEKK1 was incubated for 2 h with 6 μg of lysates from Jurkat cells stimulated with 1 μg of anti-Fas immunoglobulin M antibodies per ml for 1 h in the presence or absence of a 20 μM concentration of the caspase inhibitor Ac-YVAD-CMK (Bachem) or Z-DEVD-FMK (Enzyme Systems Products). The control lane indicates untreated in vitro-translated MEKK1. (B) In vitro-translated wild-type (wt) MEKK1 and DTVD→A mutant were incubated for 2 h with 6 μg of lysates from control Jurkat cells (−) or Jurkat cells stimulated with anti-Fas antibodies (+) as described in panel A. (C) In vitro-translated wild-type (wt) MEKK1 and DTVD→A mutant were left untreated (−) or incubated with 200 ng of purified caspase-3 (CPP32) (Pharmingen) for 1 h at 37°C (+). In each panel, the positions of full-length MEKK1 and fragments B and C are indicated.

FIG. 9

FIG. 9

The DTVD→A mutant has a reduced ability to promote DNA fragmentation in HEK293 cells. HEK293 cells were transfected with 1 μg of MEKK1.cp4, DEVE_A.cp4, or DTVD_A.cp4 and processed as described in the legend to Fig. 5. (A) Nomarski views (magnification, ×40) of HEK293 cells transfected with wild-type MEKK1 or the indicated mutants and overlaid with fluorescent staining for MEKK1 expression (red staining) and for DNA fragmentation (green staining). (B) Quantitation of the percentage of MEKK1 mutant-transfected cells that showed DNA fragmentation. The numbers in the columns indicate the number of cells transfected with the MEKK1 mutants and counted on at least four coverslips from at least two different experiments.

FIG. 10

FIG. 10

p35 inhibits ΔMEKK1-induced DNA fragmentation. HEK293 cells were transfected with 0.1 μg of MEKK1k.cp4 alone or in combination with 2 μg of p35.cp_ and stained 2 days later for MEKK1 expression and DNA fragmentation. (A) Nomarski views (magnification, ×40) of cells overlaid with fluorescent staining for MEKK1 expression (red staining) and for DNA fragmentation (green staining). (B) Quantitation of the percentage of ΔMEKK1-transfected cells that showed DNA fragmentation. The numbers in the columns indicate the number of cells expressing ΔMEKK1 and counted on four coverslips from two different experiments.

FIG. 11

FIG. 11

Lack of correlation between ERK or JNK pathway activation and MEKK1-induced DNA fragmentation. (A) HEK293 cells were transfected with 0.5 μg of the vector pcDNA_3.cp4 or with MEKK1.cp4 alone or in combination with 2 μg of CrmA.cp_ or p35.cp_. Alternatively, the cells were transfected with 2 μg of DEVE_A.cp4 or DTVD_A.cp4. The activation of ERK2, JNK1, JNK2, or JNK isoforms (JNKs) was then measured as described in Materials and Methods. EGF-R662–681, epidermal growth factor receptor peptide (residues 662 to 681). (B) Cells were transfected with 1 μg of a pCEP4-derived plasmid in which HA-tagged ΔMEKK1 was placed under the control of the metallothionein promoter (MEKK1k.MT4). Cadmium was added at the indicated concentrations at the time of serum addition in the transfection protocol. After 18 h, ΔMEKK1 expression (□), activation of JNK (GST–c-Jun1–79 phosphorylation) (◊), and the ability of ΔMEKK1 to phosphorylate SEK1 K→M (○) were measured. Data were normalized to the maximal responses.

FIG. 12

FIG. 12

Mechanistic model of MEKK1 regulation of apoptosis. See the text for details.

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